The molecular basis for the phosphorylation-dependent control of smooth muscle myosin's assembly and motor properties, and the role of these conformational transitions in vivo are examined here. We have shown by molecular genetic techniques that the native C-terminal domain of the regulatory light chain is required for phosphorylation-dependent motility, while both halves of the light chain are involved in control of assembly. Further mutagenesis will identify key sequences involved in regulating both processes. The role of the essential light chain in the regulatory switch will also be probed by analyzing the properties of myosin containing foreign or mutant light chains. Fluorescence techniques will identify phosphorylation and nucleotide-dependent interactions between the two classes of light chains. To fully understand the mechanism of regulation, it is necessary to mutate the heavy as well as the light chain. A "regulatory domain" consisting of the neck region of the myosin head and both classes of light chain will be expressed in E. coli, and as a longer term goal, the baculovirus system will be used to express a functional myosin head. The assembly properties of expressed rod fragments will be analyzed to deduce the biological role of the non-helical tail piece. Electron microscopy will show if the tail piece promotes antiparallel overlaps found between all molecules in sidepolar filaments, and critical concentration measurements will establish if the developmentally regulated isoforms of the tail piece alter myosin's assembly properties. Despite the ease with which filaments assemble and disassemble in vitro, the presence of folded myosin monomers in a cell had not been demonstrated. Through the use of a unique monoclonal antibody specific for this conformational state, we have shown by immunofluorescence and immunogold labeling that a pool of folded monomers coexists with dephosphorylated filaments in a relaxed smooth muscle cell. Digital imaging microscopy will be used to quantitate the relative amount of the folded monomer in relaxed vs. contracted tissue, and thereby determine if the monomer assembles upon activation. The broader goal of the proposed experiments is to understand how features unique to the isolated smooth muscle myosin molecule can explain the distinct mechanical properties of the smooth muscle cell.